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A stealth attack tool for preventing clinical drug resistance through a unique self-regenerating surface

Final Report Summary - BACATTACK (A stealth attack tool for preventing clinical drug resistance through a unique self-regenerating surface)

Executive Summary:
Hospital and care acquired infections (HAI) are now the fourth largest cause of death in the western world. The ECDC estimates that within Europe around 4.1 million patients (equivalent to 1 in 20 hospitalized patients) acquire infections with 37 000 patient deaths as a direct consequence of HAI. Non-fatal infections result in patients spending an over 2.500 million days in hospital. Collectively the direct and indirect cost of HAIs for the EU are estimated to be EUR 1.534 billion per annum.
Catheter-associated urinary tract infection (CAUTI) is one of the most important hospital acquired infections (HAI) and responsible for 40% of nosocomial infections. 70– 80% of these infections are attributable to use of an indwelling urethral catheter. Recent prevalence surveys report that a urinary catheter is the most common indwelling device, with 17.5% of patients in 66 European hospitals having a catheter, such as a Foley catheter. This problem is growing rapidly while resistant bacteria are spreading. The issue of resistance development is a key challenge in the conventional systemic treatment of CAUTI. Due to the high risk of developing CAUTI, 60-80% of all patients with an indwelling urinary catheter receive prophylactic antibiotics as a way to prevent the development of CAUTI. This is directly in contradiction to the European Commission’s action plan against the rising threats from antimicrobial resistance. If no solution is found, according to a U.K. government-backed report ( these threats will kill more people per year than cancer by 2050, and cost the world 100 trillion dollars annually.

The BacAttack Project has directly targeted this growing challenge. An innovative, patented carbon dioxide technology is used to create a new generation of medical devices, starting with urinary catheters, through a self-sustainable antimicrobial surface with a ‘stealth attack’ mechanism. A continuous renewal of the antimicrobial surface of the device is achieved through the release of biodegradable antimicrobial peptides (AMPs) from an interpenetrating polymer network (IPN) of hydrogel and silicone. The AMPs are stored and protected from degradation in the hydrogel network, facilitating unique controlled release over a long period. As a first line of defense, a fixed, state-of-the-art noble metal alloy coating prevents bacteria from colonizing the surface. The idea of using this unique combination of technologies, is to impart a complete long-lasting antibacterial function on medical devices, preventing selection for resistant bacteria induced by systemic treatments. This is a paradigm shift having the potential to revolutionize medical devices, as there are no similar products or technologies available despite decades of intense R&D.

The collaborative scientific and technical activities of BacAttack have resulted in identifying five main prototype candidates for further development. We have produced full-scale prototype catheters with the combination of the IPN technology, loaded with different active pharmaceutical ingredients (APIs) and coated with an effective antimicrobial metal alloy coating. The in vitro biocompatibility assessment of the four main prototypes without AMP loading demonstrated that all four candidates pass the requirements for future development of the product. These results present an in vitro pre-validation of the biocompatibility of the technology of IPN coated with “noble metal alloys”.
Three different AMPs were identified for this application of which one has potential for further development. These results show good compatibility between drugs, coatings and impregnation of the new materials. This project outcome demonstrates that the technologies of Bactiguard and BioModics are compatible, complementary and ready for exploitation in future applications. The results of the studies of the efficacy of new antimicrobial peptides with the new IPN materials are also promising. These may be candidates for a new generation of antimicrobial devices preventing infections and counteracting bacterial resistance.
These results have been disseminated widely in the form of peer-reviewed publications, presentations at conferences, meetings and at workshops which were cross disciplinary and inter project oriented as well as those open to a wider public audience and policy makers. This has resulted in a range of new commercial contacts and new collaborations to support further work with the intention of achieving commercialization of new antimicrobial devices including a Foley catheter for preventing infections.

Project Context and Objectives:
The Project Context:

In 2008 WHO launched the first global strategy for combating antimicrobial resistance. Hospital and care acquired infections (HAI) are now the fourth largest cause of death in the western world. The ECDC estimates that within Europe around 4.1 million patients (equivalent to 1 in 20 hospitalized patients) acquire infections whilst hospitalised. 37 000 patient deaths result as a direct consequence of HAIs and an additional 111 000 as an indirect consequence of HAI(Annual Epidemiological Report on Communicable Diseases in Europe 2008’) Non-fatal infections resulted in patients spending an extra 2.536 million days in hospital. Collectively the direct and indirect effects of HAIs for the EU are estimated to be EUR 1.534 billion per annum.

Catheter-associated urinary tract infection (CAUTI) is one of the most important hospital acquired infections (HAI) and responsible for 40% of nosocomial infections, leading to the most common cause of nosocomial infection in most part of the world (Mittal et al, J. Infect. Public Health, 2 (2009) 101-111). CAUTI results in morbidity, mortality, increased hospital stay and costs (Saint et al, Ann. Intern. Med., 150 (2009) 877-884). The risk of infections increases with every additional day of catheter usage. This results in a high risk of infection within a few days after insertion. The infection rate can be reduced by closed drainage and the use of catheters coated with antimicrobial agents.

The most common type of in-dwelling catheters is the Foley catheter. While Foley catheters are effective in relieving urinary retention and managing urinary incontinence, these devices provide easy access for microbes to the bladder, resulting in bacteriuria and funguria (D J Stickler, Nature Clinical Practice Uro., 11(5) (2008) 598-607). Biofilm formation is almost always associated with in-dwelling catheters and is extremely resistant to currently marketed antibiotics (Tielen et al, Int. J. Med. Microbiol. (2010)).The result can be lethal and there is a pressing need for completely biofilm-resistant devices, coatings or treatment strategies.

For decades antibacterial coatings have failed to prevent pathogenic bacteria from spreading and forming biofilms. The main reason for this is that coatings are fragile and in most cases rapidly lose their effect. This view is supported in a clinical study reported in the Lancet in which it was concluded that routine use of antimicrobial catheters could not be supported. ( The only option therefore is the development of self-regenerating surfaces that function in a controlled way during the intended period of use of the device. Recently this challenge has become realistic.

The pathogenesis of CAUTI is still unclear. How bacteria and fungi gain access to the urinary tract and bladder is important. Microorganisms colonizing the periurethral skin can enter into the bladder through the mucoid film which is formed between the epithelial surface of the urethra and the catheter. Also contamination of urine in the drainage bag may allow bacteria to access the bladder through the drainage tube and the catheter lumen. There are very few data to support the routes of microbes entry into the bladder. It is not known whether they enter the bladder along the inside or outside of the catheter, or through contamination of the tip during insertion. Microbiological methods have been used to estimate the bacteria numbers on the inside and outside of catheters removed from patients. The bacterial colonization profiles showed a mixed biofilm formed on the outside of the catheters (Barford et al, J. Urol., 180 (2008) 1522-1526), with biofilms containing 3–16 different species of bacteria. These organisms might originate from those colonizing the external urethral meatus, which were smeared up the urethra as the catheter was being inserted and then grown into a biofilm over the entire external surface of the catheter. By the use of in vitro flow models, it has been demonstrated that contamination of the tip of the catheter during insertion is a possible route by which bacteria are introduced into the bladder (Barford et al, J. Urol., 180 (2008) 1522-1526).

There are many bacterial and fungal species responsible for CAUTI. Bacteria which cause CAUTI are usually Staphylococcus epidermidis, Escherichia coli or Enterococcus faecalis. The fungal species include Candida albicans, as well as non-albicans Candida and non-Candida yeasts (J L Nates and T A Allison, The Internet Journal of Infectious Diseases, 2(1) (2002). More bacteria accumulate in the residual bladder urine in the later stages of infection including Pseudomonas aeruginosa, Proteus mirabilis, Providencia stuartii, Morganella morganii and Klebsiella pneumoniae. The microbes present in the later stages of a urinary tract infection are difficult to remove with antibiotic treatment, since biofilms offer an effective protection against antibiotics.

The issue of resistance development is a key point in the conventional systemic treatment of CAUTI. Resistance to antibiotics has developed because microbes make antibiotics themselves and have had to become resistant in order to survive naturally occurring antibiotics. When humans use an antibiotic on a huge scale, sensitive bacteria are killed but naturally resistant ones survive and multiply. After a few years, these resistant mutants have spread worldwide and eventually are so common that the antibiotic becomes redundant. Bacteria have many different ways of becoming resistant, for example they can transfer resistance genes between distinct species of bacteria. Hence, the launch of every antibiotic has been and will be followed by resistance in the targeted bacteria. Therefore, there has been a constant need to develop new agents to keep up with the acquisition of resistance among pathogenic bacteria. However, now bacteria are developing resistance faster than the world can make new antibiotics to deal with them. New antifungal drugs for urinary Candida infections are also needed (D W Denning and W W Hope, Trends in Microbiology, 18(5) (2010) 195-204).

Development of tools to control microbial biofilms is a problem due to bacteria multi drug resistance and a lack of new strategies. It may be relevant to ask the question: Are we losing the battle? This is still an open question however the European Centre of Disease Prevention and Control (ECDC) has stated that: ‘if this wave of antibiotic resistance gets over us, we will not be able to do organ transplants, hip replacements, cancer chemotherapy, intensive care and neonatal care for premature babies (Paul Fiddian, ‘Warning on over-prescribing antibiotics’, Pharmaceutical News, November 2009)’. The BacAttack project combines several unique approaches into a platform capable of merging existing tools with new promising tools specifically designed to control the microbial biofilms problem and alleviate problems resulting from clinical drug resistance.

Overall Concept and Objectives:

The BacAttack proposal will focus on technologies based around new hybrid materials and advances within Anti Microbial Peptides (AMPs) and on integrating them into a material with a self-regenerative surface. By focusing on both material and drug, the tools developed for preventing biofilms will result in a generic methodology. The goal of the project is to build a platform for future medical combination devices that is cost-effective and for the first time highly functional. The platform will build upon existing antimicrobial tools to maximize the use of existing know-how and infrastructure. The platform will use low throughput prototyping equipment to develop the materials and the integration for the platform. The ability to integrate the platform into a complete product will be demonstrated through a product prototype. The objective of the project is to introduce a paradigm shift for preventing infections and bacterial multi-drug resistance in a highly effective, unprecedented and environmentally friendly way.

The BacAttack Project Concept

New Materials Technologies based on Supercritical Carbon Dioxide:
An innovative patented carbon dioxide technology (J Karthaeuser, WO 2005/003237A1) will be used to create a new generation of medical devices through a self-sustainable antimicrobial surface with a ‘stealth attack’ mechanism as follows. A continuous renewal of the antimicrobial surface is achieved through the release of environmentally friendly biodegradable antimicrobial peptides (AMPs) from an interpenetrating polymer network (IPN) of hydrogel and silicone. The AMPs are stored in the hydrogel network which will facilitate unique controlled release over a long period. The network will also protect the unreleased AMP from degradation. As a first line of defence, a fixed noble metal alloy surface will be applied to prevent bacteria from colonizing the surface. This two-fold antibacterial action can subsequently be applied to a range of medical devices. The technology will give coatings on medical devices a complete long-lasting antibacterial function and prevent selection for resistant bacteria induced by systemic treatments. This generic tool set will also be highly effective against pathogenic fungi and over all provide a major step towards improvement and maintenance of citizen welfare. This is a paradigm shift, which has the potential to revolutionise medical devices as there are no similar products or technologies available despite decades of intense R&D.

Stealth Replaces ‘Blitzkrieg’:
Instead of a systemic ‘blitzkrieg’ approach, the technology described in this project provides a local ‘stealth attack’ against bacterial and fungal infections, by utilizing a combination of material and microbial strategies. In the first instance, urinary catheters will be used as a test bed for the new technology. However, the ultimate aim of the project partners is to widen the scope and include other medical devices, such as stents, wound dressings and contact lenses.

The BacAttack project will generate a paradigm shift by use of silicone rubber as a platform for hydrogel impregnations with groundbreaking drug delivery capabilities.

The BacAttack Project Objectives:
The specific and measurable objectives to be achieved within the project are:

Objective 1: To develop add-on material treatments for silicone devices based on supercritical carbon dioxide (scCO2) technology to produce new hydrogel silicone IPN materials.
Objective 2: To develop hydrogel silicone IPN materials with tailored release properties.
Objective 3: To incorporate antimicrobial peptides in the IPN materials for long term release.
Objective 4: To investigate broad spectrum antimicrobial peptides.
Objective 5: To combine hydrogel silicone IPN materials with noble metal alloy coatings that reduces bacteria adhesion and colonization.
Objective 6: To investigate the materials regarding performance in vitro and biocompatibility.
Objective 7: Prototype demonstration.

The BacAttack project contains two primary technologies, IPNs and AMPs, these will be combined with a third, the Bactiguard noble metal alloy-coating, which is the current state-of-the-art solution for antimicrobial catheters. BioModics is the provider of the IPN technology, based on impregnation. In December 2009 BioModics presented the tool box strategy at the Eurecan European Venture Contest after passing a number of regional events in Europe with 800 European companies. An international jury selected BioModics as the overall winner in all categories comprising life sciences, ICT and clean technology. It was stated: ‘BioModics have demonstrated the excellence of Europe’s drive to succeed in high tech’. Novozymes and the Weismann Institute are providers of the AMP technology. They are world-leaders in this area complementing each other. Bactiguard is a provider of state-of-the-art anti-infectious noble metal based technology to one of the world’s largest providers of medical devices. The project seeks to create a hybrid solution consisting of the Bactiguard coating and AMP-loaded IPNs to create self-regenerating surfaces with a two-fold antimicrobial action.

The consortium has a clear research plan: The hybrid solution is to be used for preventing microbial infections associated with medical devices. Moreover, this approach provides an efficient paradigm to minimize development of bacterial resistance with a local and efficient delivery of new AMPs.

Calvin Kunin, in his editorial in the New England Journal of Medicine asked the question: ‘Can we build a better catheter?’ The ultimate success criterion of the BacAttack project will be to produce a fully functional prototype catheter that will prevent CAUTI whilst providing a solution preventing the problem of resistance development in bacteria.

Project Results:
BacAttack Work Plan:

To achieve the project objectives of the BacAttack project our work plan included eight interconnected work packages (WPs). Each WP had associated deliverables which provided details of the work carried out. Progress of the project was determined through achievement of the work packages’ contribution to reaching milestones set out at the start of the project. The WPs and their interrelation in contributing to the goals and objectives of the project are described briefly below:

In WP1, the development of IPN materials will be accomplished by optimization of supercritical carbon dioxide (scCO2) technology to impregnate silicone with hydrophilic hydrogels. By using scCO2 as an auxiliary solvent, the silicone will absorb the hydrophilic monomer which is subsequently polymerized and optionally cross-linked. The result is an IPN consisting of silicone and cross-linked hydrogel. The work involves optimization of parameters for the scCO2 process, selection of chemistry for the hydrogel polymerization as well as combination with Bactiguard (registered trade mark) noble metal alloy coated catheter materials. This WP will provide input for WPs 3 and 4, and contribute to MS1.

WP2 concerns research and product development of AMPs and ultrashort lipopeptides to achieve broad spectrum antibacterial effect without induction of resistance. It is divided into two tasks. Task 1 concerns research on two novel families of AMPs: 12-15 amino acids long peptides and ultra short lipopeptides (2-4 amino acids linked to a fatty acid) that will be modified to be incorporated in the IPN materials. Task 2 involves recombinant peptide production development, identification of suitable AMP sequences, and process scale-up. The outcome of WP2 will be selected AMP and lipopeptide candidates to be used in
WP3 and a documentation of the scaling up of the processes for producing sufficient quantities for future commercialization. WP2 will give input to WP3 for incorporation into the IPN, to WP6 for prototype production and contribute to milestones MS2 and MS3.

The aim of WP3 is to incorporate AMPs selected from WP2 into the new silicone hydrogel IPNs combined with Bactiguard noble metal alloy coating developed in WP1. This WP is divided into three tasks. Task 1 concerns incorporation of selected peptides from WP2 in the IPN by loading from a solution or by incorporation during hydrogel synthesis. Task 2 concerns physical, chemical and mechanical characterisation of the integrated product as well as investigation of release profiles of AMPs. WI in WP2 will undertake assessment of the stability of the AMPs after loading and release by fluorescence studies. In task 3 sterilisation routes for the combination product will be investigated and shelf life studies will be initiated. The WP3 will combine knowledge generated in WPs 1 and 2 and the outcome of WP3 will be selected AMP-loaded IPN materials combined with Bactiguard coating with documented long term release of AMPs and maintained stability after sterilisation. WP3 will give input to WP6 and contribute to reaching MS2, MS3 and MS4.

WP4 concerns in vitro assessments of the materials for regulatory issues. The biocompatibility of starting materials used in WP 1 will be tested. Selected combination products from WP3 as well as the prototype developed in WP6 will be assessed regarding efficiency, biocompatibility (cytotoxicity, genotoxicity and urinary tract cells behavior in contact with the material). The milestones of WP4 will be a documentation of the biocompatibility, in vitro efficiency and regulatory issues. WP 4 will give input to WPs 1, 2, 3, 5 and 6 and contribute to MS1, MS4 and MS6.

WP5 will study the performance of the catheters hybrid materials derived from WP3 and the final prototypes form WP6 against microorganism adhesion, colonisation and biofilm formation using microbiological methods. The long-term effects of the hybrid material such as release of AMPs will be tested using the same methods. The bacteria used for this WP will be pathogens which usually associated with CAUTI such as Escherichia coli (NCTC 9434), Pseudomonas aeruginosa (NCTC 6751) and Enterococcus spp. Resistant strains will be included. Most importantly, some Gram-negative and Gram-positive bacteria (100 strains) isolated from patients with urinary tract infections and CAUTI will be used to test the clinical significance of these coating materials and coated catheters. This WP will give input to WPs 1,2,3 and 6 and contribute to milestones MS4 and MS6.

In WP6 the IPN production setup will be scaled up to fit a full size catheter based on the knowledge generated in WP1-WP3. Materials selected on the basis of the results form WP3-5 will be used for production of the prototype. The outcome of WP6 will be a demonstration device; ‘catheter prototypes with self-regenerating surfaces capable of long term antimicrobial effect by controlled release of AMPs combined with a noble metal alloy’. WP6 will give input to WP4 and WP5 and the milestones MS5, MS6 and MS7.

Project management and coordination of consortium will be carried out throughout the project lifetime (WP7). Dissemination of results, protection of IPR and other innovation related activities will also be carried out throughout the project period (WP8). Activities in WP7 will facilitate smooth running of the project, timely generation of reports and arbitration of conflicts, whilst activities in WP8 will address the dissemination of project results and intellectual property management (an agreement between the partners will be signed).

The milestones set at the start of the project were:
MS1: Selection of biocompatible IPN material combinations to be used in WP3 and WP5
MS2: Selection of broad spectrum AMPs and lipopeptides to be used in WP3 and WP6
MS3: AMP-loaded IPN materials selected for combination with Bactiguard coating
MS4: Establishment of a viable sterilisation procedure to be used for the combined material.
MS5: Prototype produced and production route verified.
MS6: Completion of biocompatibility, in vitro safety and efficiency studies and in vivo safety studies.
MS7: Demonstration of proof of concept

WP 1 IPN Development.
The objectives of this work package were to investigate the IPN technology and select relevant materials and processing conditions for producing silicone/hydrogel compositions to be applied under WP3.
This work package addresses the development of IPN materials of silicone and hydrogel using supercritical carbon dioxide (scCO2) technology, which gives a unique possibility to implement IPNs as an add-on to existing silicone devices. By using scCO2 as an auxiliary solvent, the silicone will absorb the hydrophilic monomer which is subsequently polymerized and optionally cross-linked. The result is an IPN consisting of silicone and cross-linked hydrogel capable of long term release of active ingredients. The IPN will furthermore be combined with the Bactiguard (registered trade mark) noble metal alloy coating. The technological platform for the development, the synthesis, and the characterization of the materials will be optimized with respect to the catheter application. The Partners involved in this work package were BioModics (BM), Bactiguard (BG) and Danish Technological Institute (DTI). DTI led this WP.

During the first 12 months of the project the synthesis protocols were optimized in order to control batch homogeneity and reproducibility between batches. The hydrogel content and the degree of cross-linking were varied in order to control the IPN water uptake.

Manually controlled, small-scale systems were initially used to produce IPN materials by scCO2 technology. Two types of silicones, relevant for urinary catheters were identified by BG, BM and DTI. BG supplied the silicone materials for further studies. Non-coated catheters and catheter tips have also been delivered by BG. Flat samples (discs or rectangular shape), catheter pieces and tips were successfully treated to create IPNs. Both types of silicones allowed monomers to be impregnated and subsequently polymerized inside the pores of the silicone.

Different hydrogel formulations were incorporated into the silicone matrix. Various formulations with co-monomers were investigated by DTI and BM. A formulation that resulted in a very hydrophilic hydrogel was developed by DTI. Studies resulted in a high degree of success to optimize the process for the production of IPN’s. Selected IPN materials were characterized by physical, chemical, mechanical and imaging techniques. These included swelling performance of IPNs in water, salt solutions and urine broth. Chemical composition studies included FT-IR, TGA and DSC. Florescence techniques were used to study release properties of antimicrobial and model substances. Micro-CT scanning was a powerful tool to visualize the hydrogel channels in the IPN matrix. Characterization of metal surface concentration and release of metals from pre- and post-treated Bactiguard-coated IPN samples has been made by BG.

A new, state-of-the-art supercritical CO2 system was purchased by DTI and was successfully installed which enabled production of large amounts of test samples needed for biocompatibility testing under WP4 with batch-to-batch and sample-to-sample reproducibility. Samples loaded with a model active pharmaceutical ingredient (API) as well as antimicrobial peptides (AMPs) including Novocidin and Plectasin from Novozymes (NZ) and newly developed AMPs from Weizmann Institute (WI) were sent to St. George’s University of London (SGUL) for bacterial testing under WP5.

Combining the IPN materials with the Bactiguard coating technology was achieved. SEM of DTI samples evaluated by BG showed a pore size of 5 micrometers and a nanostructure. Results from BG’s Ahearn tests showed a 100% reduction of bacteria on IPNs combined with their coating.

Initial cytotoxicity studies from LEMI indicated that the sterilization technique selected may influence the biocompatibility of the materials. Cleaning techniques were optimized to resolve this problem.
WP1 started on M1 and originally had its end-point in M12. However, it was decided to extend this task to run in parallel with WP3 as these work packages were interdependent. Apart from achieving the goals and objectives of this task, problems encountered with equipment, purchase of additional chambers and spare-parts and other consumables associated with running the scCO2 process and equipment were resolved with the support of WP1.

WP2 Antimicrobial Peptides - Identification and Development
The overall objectives of WP2 for WI were to design an antimicrobial peptide (AMP) against a broad spectrum of pathogens (both Gram-positive and Gram-negative bacteria), with the following properties: (i) anti-septis due to its ability to neutralize LPS and LTA (the cell wall components of Gram-positive and Gram-negative bacteria, respectively) mediated activation of immune cells, (ii) low toxicity and ability to inhibit biofilm formation, as well as to destroy established biofilms, (iii) ability to overcome bacterial resistance via various mechanisms, (iv) low cost, (v) long shelf life, and most importantly they should be reversibly loaded into the IPN with high efficiency and biological function, and with long time release from the IPN. The focus for NZ was on broad-spectrum AMPs of 15-25 amino acids, mainly of the alpha-helical class. These peptides are broadly active on gram-positive and gram-negative bacteria as well as against certain yeasts and fungi, kill in minutes and have a low propensity for resistance development. Existing peptides will be evaluated and their compatibility with IPN and catheters tested. Also, new peptide variants will be constructed that have optimal features for this application – e.g. release profiles, stability and compatibility. In parallel, recombinant production systems will be established. The partners involved in the activities of this WP were Novozymes (NZ) and Weizmann Institute (WI).WI led WP2.

Peptides parameters required for toxicity and neutralization of endotoxin mediated sepsis:
WI synthesized a series of native and de-novo designed peptides. The new peptides were all derived from a well-known peptide, MSI-78, used as a model peptide. It is a 22 amino acids antimicrobial peptide (megainine2 analog) with a broad spectrum of activity against Gram-positive and Gram-negative bacteria and Candida albicans. All the analogs have the same amino acids composition but with varying order of the amino acids. Our main goal was to separate between their antimicrobial activity while keeping reduced toxicity towards mammalian cells. Their activity in neutralizing endotoxins by reducing cytokine secretion from RAW264.7 (macrophages) cell line was analyzed. In addition several biophysical methods were used in order to determine their secondary structure in solution and how this structure affects their activity. Further data was collected on the location of the peptides in/on the cells by using confocal microscopy and a state of the art image stream analysis. The WT peptide was designed to target the membrane which retained it more toxic. Studies on its mechanism of action revealed that it coats the membrane, in comparison to some of the derivatives that had high activity in neutralizing endotoxins, both LPS and LTA (Gram negative and Gram positive, respectively). In contrast, their anti-microbial activity was very weak. They adopted a beta structure when bound to the endotoxin compared to the alpha helical structure of the WT.
Overall, this study revealed the importance of the peptide’s parameters such as hydrophobicity, order of the amino acids and secondary structure, in both its antimicrobial and endotoxin neutralization activity.

Peptides parameters required for prevention and degradation of pre-formed biofilms.
For this study, WI designed and synthesized a family of 15 mer peptides composed of Lysines and Leucines that differ in their charge distribution and hydrophobicity in order to maximize their activity. We also altered five amino acids to their D- enantiomers to reduce toxicity and enzymatic degradation. WI studies showed that all peptides that possess antimicrobial activity against planktonic bacteria were also active against biofilm forming bacteria. These AMPs prevented biofilm formation in their minimal inhibitory concentration (MIC) by reducing planktonic bacteria growth and killing it. They also reduced biofilm formation using nM concentrations by reducing the bacterial adhesion to a surface. It was revealed that the mechanism of these peptides is coating of the un-organic surface, bacterial cell or both.
Importantly, biofilm degradation by these peptides is achieved via two possible mechanisms: (i) killing of biofilm embedded bacteria and their detachment from the biofilm milieu, (ii) detachment of live bacteria from the biofilm milieu (by non-bactericidal peptides). Fluorescent spectroscopy studies revealed that the active peptides are equally distributed all over the biofilm layers. WI also found correlation between the peptides activity and their concentration in the mature biofilm. The peptides that were found in high concentration in the biofilm were also more active.
It was concluded that L-to-D-amino acid substitution contributes to antibacterial activity. It reduced self-assembly, allows structural flexibility, and reduces enzymatic degradation and hemolysis. These peptides are highly active against all stages of biofilm formation and act in a variety of parallel mechanisms.

To address the question of resistance development by bacteria to antimicrobial peptides, WI investigated two bacteria; pseudomonas aeruginosa and Salmonella as well as a large number of peptides to test whether they develop resistance towards them. The main resistance mechanism against AMPs in Gram negative bacteria is lipopolysaccharide (LPS) alternation, which is activated by two separate two component systems (TCS). WI tested whether activation of the TCS is reflected in more resistant bacteria against two of our most potent peptides. The showed that the MICs against 2 selected peptides (AMP1L and AMP1D) remain the same even when activating the TCS, which suggests that no resistance is achieved.

In the case of Salmonella resistance to antimicrobial peptides, WI aimed to evaluate whether peptide characteristics can affect the evolved mechanism. 9 resistance lines of bacteria to 9 peptides with distinct properties were generated. Experimental evolution was used by growing WT bacteria in sub inhibitory concentration of peptides, while increasing this concentration by 50% for 30 passages, each for 24hr. Results showed that for some of the peptides resistance evolved while for some this was not the case. R4 (which became WI’s lead compound) and C8-K5L7, synthetic peptides from WI, did not induce resistance in our study. However, for some peptides resistance was induced and the bacteria exhibited an increase in the MIC value. Resistance line 1 (RL1) was selected to a particular peptide (4D-K5L7) and showed a dramatic increase in the MIC value in comparison to the WT. Therefore investigations on RL1 biophysical and biochemical properties were carried out in order to interpret its mechanism of resistance.

It was found that RL1 show cross resistance to mostly short, high charge density, and low hydrophobicity peptides. RL1 was more sensitive than the WT to peptides with opposite physical properties. This could indicate that the mechanism of resistance involves modifications on the bacterial surface. Observation of the bacterial cells in Transmission Electron Microscopy (TEM) did not reveal any differences in the cell wall or the membrane structure. However, we noticed that RL1 contained a sub-population of elongated, multinucleated cells. This phenotype was remarkable since it was never reported before in association with resistance to AMPs.
Further investigation of the length property showed that elongation is dependent on the growth stage. While elongation is maximal in early stages of growth, it decreased in advanced stages. Moreover, when we segregate RL1 population to short and long cells via FACS and grow them separately, they both maintained their potential to form a heterogeneous population. Interestingly, RL1 bacteria showed a defect in swarming motility, which to our knowledge, is a novel resistance associated phenotype.

In summary, results from WI suggest that there are peptides that bacteria struggle to gain resistance to. Therefore, these peptides are good candidates for future development of antibacterial drugs. On the other hand, RL1’s cross resistance and other novel phenotypes might point to a novel and unreported mechanism of resistance. The information gathered in this complex study contributed significantly to select R4 as the lead peptide for IPN loading.

NZ conducted the following work for activity testing of antimicrobial peptides. The AMPs of relevance to the project goal were identified: Peptides with activity against pathogenic strains of E. coli (urinary tract infections) and S. aureus (catheter infections). Profiling of the antimicrobial activity was performed by MIC (minimal inhibitory concentration) studies against a range of strains of E. coli and S. aureus. Novicidin was selected as the test compound against Gram-negative urinary tract infections and was tested against 2 type strains of E. coli and 38 clinical isolates, all known to be ampicillin/cefazolin-resistant. The MIC values ranged from 0.5-4 for all isolates, except four with had MICs of 8-16.

As a second AMP, Plectasin, NZ2114, was selected as the test compound against S. aureus and other Gram-positives as it has shown strong activity against Methicillin-resistant staphylococci (MRSA) and streptococci. In a MIC testing of 32 strains of S. aureus, of which 13 were MRSA, the MIC values ranged from 0.5 – 4. MIC testing was performed in accordance to the general guidelines provided by NCCLS: MIC determination: M7-A6, vol. 20, No. 2: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Sixth Edition, Jan 2003.
Novicidin and Plectasin were tested for toxicity in vitro in hemolysis (RBC), cytotoxicity (NRU) and skin irritation (Episkin) assays.
▪ Results showed that Plectasin was non-hemolytic in concentrations up to > 512-1024 ug/ml. HC50 of NZ1024: > 282 ug/mL
▪ Cytotoxicity assay was carried out in mouse fibroblast cell line. Results showed no signs of cytotoxicity from Plectasin with NRU50 values from 500-2000 ug/mL. Novicidin was slightly cytotoxic with NRU 50 values of 169-320 ug/mL.
▪ Episkin is an assay which is OECD/ECVAM validated for irritation/corrosion studies. It consisists of human derived adult keratinocytes grown for 13 days for differentiation into stratified epidermis model. Results showed that neither of the AMPs were irritant to human skin. Novicidin (10 mg/mL): 92 %. Plectasin (10 mg/ml): 93 %

NZ has applied different methods for recombinant expression of AMPs in order to produce significant amounts of peptide for testing. The challenges for expression lie in the dilemma of producing an antimicrobial peptide in a microbial host organism. Furthermore, the un-structured peptides (α-helical and amino acid enriched) may be very susceptible to proteolytic degradation the fermentation broth. Expression level in g/L for small molecules may be difficult to achieve due to limitation in the secretory pathway (number of molecules not grams).
For alpha-helical peptide (Novicidin) different techniques have been tested: Multimer expression to E. coli inclusion bodies, yeast systems and multimer expression in Bacillus and Aspergillus with down-stream process of dissolving, maturation and capture of peptide.
Multimer expression in E. coli showed most promising results for larger scale production while yields were very low when applying other techniques. This process resulted in a “proof-of-concept” set-up with low yields of Novicidin. For full scale production further optimization of down-stream process would be needed. The process is labor intensive and furthermore, the AMP seemed to be unstable and not active in urine (see release studies). A large amount of Novicidin was also produced by chemical synthesis.

For disulphide-stabilized AMP (Plectasin) monomer expression in an Aspergillus host gave significant yields. Production and expression as described by Mygind et al. (Nature, 2005). This process was easy to handle and resulted in a commercially applicable process. The resulting peptide was highly stable.
Release studies: NZ has tested the release of Plectasin and Novicidin from impregnated silica discs (supplied by DTI). Release was tested from discs into radial diffusion agar (RDA) plates:
- Release into 0.9% NaCl and urea broth (Sigma)
- RDA with Staphylococcus carnosus ,S. aureus and E. coli

LC-MS quantification
Results showed that S. carnosus, S. aureus but not E. coli are sensitive to Plectasin.
Plectasin is released into 0.9% NaCl including into the last sample representing release from day 76-90.
Plectasin was released into urea until after day 48 (release from day 41-48).
Plectasin was detected by LC-MS in both 0.9% NaCl and in artificial urine.
Release into 0.9% NaCl was detected by LC-MS until after day 20, representing release from day 13-20.
Release into urine was only detected by LC-MS until after day 13, representing release from 6-13
All three test strains S. carnosus, E. coli and S. aureus are sensitive to Novicidin.
Novicidin is released into 0.9% NaCl including into the last sample collected representing release from day 76-90.
No release of Novicidin into urea from impregnated discs was observed.
LC-MS analysis detected Novicidin in 0.9% NaCl until after the sample collected day 27 representing release from the disc day 20-27.
Release of Novicidin into artificial urine was not detectable by LC-MS.

Delivery of antimicrobial peptides: Several grams of both AMPs (Plectasin and Novicidin) has been supplied to project partners during the project period.

WP3 Integration of IPN materials with antimicrobial strategies. Partners involved in this work package were BM, DTI, WI, NZ, BG.
The main objective of this work package was to incorporate AMPs into the new silicone hydrogel IPNs capable of long term release, and to combine the loaded IPN material with the Bactiguard noble metal alloy coating (pre- and post-treated). Besides AMPs, other active ingredients with antimicrobial functions may also be assessed in order to fully characterize the loading capability and release properties of the new materials. Physico-chemical characterization of material properties will also be assessed. WP3 was led by BM.

Task 3.1 - Integration of IPN Materials
Subtask 3.1.1 - Loading of IPNs with Selected AMPs and Active Ingredients
During the first 18 months, the partners performed initial trials with different model drugs and AMPs to identify suitable candidates for further studies. IPNs where made using the shaft of BactiGuard B.I.P. silicone Foley catheter. As a model material, discs were cut from an extruded band made of the same silicone material as the BIP catheter. On these two materials (shafts and discs), various hydrogels were evaluated by BioModics using homo- and copolymers of PHEMA, PEGMEA, PMAA. DTI developed a hydrogel based on poly(ethylene glycol) methacrylate (PEGMA) to obtain a soft IPN with a high water uptake and mechanical flexibility.
Initially, high-pressure impregnation wasinvestigated for loading the IPNs with active pharmaceutical ingredients (API’s) to achieve antimicrobial properties. IPN discs with varying PHEMA content were loaded with a wide range of antimicrobials including chlorhexidine, gentamycin, meropenem, nitrofurantoin, novicidin, plectacin, silver lactate and trimethoprim under high pressure. Even though the IPNs loaded with API’s demonstrated anti-microbial effects when tested on agar-plates with E.coli there was a large sample-to-sample variation.
Therefore, conventional loading methods e.g. passive diffusion were focused on, where the non-swollen IPN is placed in a solvent containing the drug. The solvent causes the IPN to swell whilst the drug migrates with the solvent into the IPN reaching the interior of the matrix. The model drugs and APIs loaded by this method were e.g. acid orange, cetylpyridinium chloride, nitrofurantoin, PA-KKK and PA-KLK.
The intermolecular interactions between hydrogel, silicone, drug and solvent were evaluated in terms of loading and release of the APIs. The following conclusions were reached based on studies to optimise the loading and release properties of the system:
- Properties of the drug e.g. the lipophilicity of the drug influences loading and release kinetics amounts of drug.
- Effect of loading media. Release profiles can be tuned by choice of loading media and loading concentration.
- Effect of hydrogel type. The affinity between drug and hydrogel (and hence the release profile) depends on the hydrogel system.
- Effect of hydrogel content. An increased hydrogel content allows for a higher loading and release of the API
- Media effects. Release and efficacy of the API is influenced by the media into which it is released
- Loading procedures influence the final loading of the IPNs. Changing some loading parameters like temperature, “preloading” with solvent, pressure impregnation and double loading have been investigated.
Furthermore, a thermodynamic model that described the loading process was developed.

Subtask 3.1.2 Combination of the Bactiguard Coating with and without AMP Loaded IPN Material
IPN samples was coated using BGs proprietary noble metal coating. The BG coated IPN materials were characterized with respect to noble metal surface concentration, metal release, and microbiological studies.
The surface concentration of silver was measured by Atomic Absorption Spectroscopy (AAS) analysis (Air-Acetylene flame). Initially, IPN samples post-treated with BG’s surface coating showed a higher amount of silver on the surface than the commercially available BG coated silicone catheter. During the project, BG optimized their coating on IPN treated material achieving the same noble metal surface concentration and extremely low metal release as the commercially available Bactiguard Infection Protection (BIP) coating used on Foley catheters.
Ahearn tests for antimicrobial efficacy were performed to evaluate the primary adhesion of bacteria on to uncoated IPNs and the BG coated IPNs. The procedure for performing the Ahearn test was done according to BG’s Standard Operating Procedures. For this investigation BG used the bacteria Pseudomonas aeruginosa ATCC 6751. The bacterial adhesion was analyzed after soaking in artificial urine for 0, 1, 5, 10, and 15 days. The BG coated IPN samples showed a 100% reduction of bacterial adhesion and biofilm formation. Interesting results were also obtained for the IPN material from DTI (not coated with the BG coating) that showed a measurable reduction in bacterial adhesion.

Task 3.2 – Characterisation of Integrated Product (BM, DTI, BG, WI, NZ) M12-M45
Subtask 3.2.1 - Physical, Chemical and Mechanical Characterization
The physico-chemical properties (hydrogel content, degree of swelling, FTIR, TGA, contact angle measurements, loading homogeneity and efficacy (LSCM)) and mechanical characteristics of produced IPN samples have been analyzed throughout the project. These studies have helped us to further develop and optimize the IPN material to the full size prototypes that were produced in WP6 towards the end of the project. The characterization data will also contribute towards the documentation for CE-marking of future products.
BM loaded IPN samples of different types of silicone and hydrogel with different fluorescent/non-fluorescent dyes to document drug distribution and loading efficiency, under varying loading conditions e.g. varying the host polymer (silicone elastomer), guest polymer (hydrogel), loading solvent and loading time.
The results indicated that there is a critical value for the free volume in the IPN matrix. Under the critical value of the free volume, a very limited amount of drug can be impregnated into the IPN. Furthermore, various parameters influence the free volume in the IPN e.g. the chemistry of the hydrogel material, the hydrogel content and the choice of loading solvent. Results showed that an IPN with PHEMA can be loaded homogeneously with fluorescein in ethanol during a loading period of three days. For some hydrogels, ethanol acts as a better loading media for IPNs than water by increasing the degree of swelling, hence increased free volume, during loading. Results also showed that the amount of fluorescein loaded into the IPN increases with an increase in the hydrogel content.

Physical dimensions of samples prior and post IPN treatment and swelling in different solvents were measured by DTI. The results confirmed that the IPN treatment was homogenous through the sample thickness.

Subtask 3.2.2 - Investigation of Release Profiles and Stability of AMPs after Release
Due to the initial limited access to antimicrobial peptides, model drugs and molecules were used for the initial release testing. DTI used Acid Orange 8 (AO8) as a model drug that is hydrophilic and can be easily detected using UV-VIS. The release studies of model drugs Acid Orange 8 and cetylpyridinium chloride by DTI were carried out under static conditions, where fresh solution was exchanged during regular time intervals. DTI also conducted nitrofurantoin solubility, loading by solvent mixtures and release tests in aqueous media. Nitrofurantoin loaded IPN and controls were sterilized and sent for testing at LEMI and SGUL. Nitrofurantoin loaded IPN samples were also produced for optimization of the combination with the BG coating by post-coating.

BM initially used two model drugs, methylene blue and trimethoprim, as well as methylene blue for their release tests. Release profiles of different types of IPNs were analysed and BM found that it was possible to model the release profiles and diffusion coefficients could be estimated.
Following optimization of IPNs produced by BM and DTI, various loading and efficacy studies were carried out to study the antimicrobial effects of API including two antimicrobial peptides, Novocidin and Plectisin from NZ and the R4 peptide from WI. The APIs loaded into the prototypes, as a result of the activities under WP3, included Nitrofurazone, Colistin Sulphate (CS) and the R4 peptide. The release studies, antimicrobial activity and biocompatibility evaluations are reported under WP2, WP4 and WP5.
WI carried out the tests for the release of R4-A from IPN discs using HPLC. The loading of the IPN discs had taken place at room temperature. A constant release of R4-A from the discs was found. However, the release levels out after about 70 h where about 70 % of the R4-A was released. The tests performed by WI were carried out without changing the media. Therefore saturation of R4-A probably occurs after a certain period of time in the media and the release will be affected. The tests should ideally be repeated with continued change of media. Future tests should also be performed using artificial urine.
Studies carried out by SGUL on samples received from WI and DTI showed that the antimicrobial activity of the R4-A peptide was seriously impaired when subjected to a temperature of 50 °C during loading.
Sterilization of IPN samples: Initial sterilization of IPN samples sent for biocompatibility testing indicated cytotoxicity. However, after optimizing cleaning procedures, biocompatibility testing indicated that EtO sterilization, currently used by BG on their commercial BIP silicone catheters, could also be safely applied with the IPN treated catheters.
Shelf life and Ageing studies: Mechanical tests were performed on untreated 12Fr silicone catheter tubes of 7 cm length and DTI’s prototype IPN after exposure to ageing tests under the following conditions. Series 1 was exposed to ageing in simulated physiological conditions in a urine flow system for four weeks. Series 2 was exposed to accelerated ageing (shelf life testing) in oven at 60°C for four weeks. The selected time points were 0 (start), 1, 2 and 4 weeks (end). The accelerated ageing tests were performed according to ASTM F1980 “Standard Guide for Accelerated Aging of Sterile Barrier Systems for Medical Devices” and represented a shelf life of one year.
The tensile testing was performed with reference to standards ISO527 “Plastics - Determination of tensile properties” and ASTM D 638 “Standard Test Method for Tensile Properties of Plastics“. At selected time points, triplicates of dumbbell specimens or catheter tubes were removed from the exposure test and the mechanical parameters stress (MPa), strain (%) and E-modulus (MPa) were determined by tensile testing. Results indicated that for ageing under simulated physiological conditions, Series 1, the mechanical properties stay relatively constant for both the standard silicone catheter and the DTI prototypes.
For samples tested under the accelerated ageing conditions, the properties of the standard silicone catheter showed little change as expected. However, there were significant changes for the IPN prototypes, due to a possible thermal degradation of the hydrogel in the material. The accelerated ageing study will have to be repeated for conditions appropriate for the IPN material.

WP4 Biocompatibility, in vitro and in vivo Assessment for Safety and Regulatory Issues
This work package focuses on in vitro assessments of the materials to obtain documentation according to standardised methods as in-put for future CE-marking. The biocompatibility of the starting materials used in WP 1 will be tested. Selected combination products from WP3 as well as the prototype developed in WP6 will be assessed regarding efficiency, biocompatibility (cytotoxicity, genotoxicity and urinary tract cells behaviour in contact with the material). The work carried will be complementary to the in vitro testing by models simulating clinical use as further defined under WP5. This WP was led by LEMI

During the BacAttack project LEMI has:
➢ developed and validated in vitro test techniques for new antibacterial device development :
− antibacterial efficiency ;
− use of urothelial cells for biocompatibility assessment ;
− genotoxic assessment (micronucleus) using TK6 cells.

➢ developed research and testing concerning :
− Task 4.1. Antibacterial efficiency testing (biofunctionality) of materials selected in WP3.
− Task 4.2 In vitro assessment of biocompatibility of materials selected in WP3 which includes: cytotoxicity testing, cytocompatibility assessment using human urothelial cells and human monocytes-macrophages.
− Task 4.3. In vitro assessment of biocompatibility of prototypes selected in WP6: cytotoxicity testing, cytocompatibility testing, genotoxicity testing.

1. Antibacterial efficiency testing (biofunctionality) of materials selected in WP3 (Task 4.1).

To be effective a “material” must control bacterial adhesion and proliferation. The following test systems are tested at LEMI to be used in efficiency testing of materials arising from WP3 :
− Escherichia coli (ATCC-35218) (Gram-negative)
− Staphylococcus epidermidis (ATCC-35984) (Gram-positive)
− Staphylococcus aureus (ATCC-6538) (Gram-positive)
− Pseudomonas aeruginosa (ATCC-27853) (Gram-negative)
− Candida albicans (ATCC-10231)

Samples tested:
In total, 14 samples produced under WP3 and received from BM and DTI were tested. These included IPNs produced by BM consisting of two different silicones combined with 2 different two types of hydrogels. In total 5 different samples from BM were tested. DTI provided samples with one silicone material provided by BG an one optimized hydrogel that had been subjected to various cleaning procedures and loaded with Nitrofurantoin as well as Plectasin and Novocidin. An unloaded IPN material was provided as the reference. In total, 9 different samples from DTI were tested.

Qualitative results: Of the 14 materials tested, 9 samples (7 provided by DTI and 2 from BM) showed a reduction, or an absence, of bacteria attachment and adhesion.
Quantitative results: The quantitative method highlighted the following three test materials from DTI: IPN loaded with Nitrofurantoin, Plectasin and Novocidin, respectively. For the Nitrofurantoin and Plectasin loaded materials, bacterial adhesion is decreased by 3 to 10 times for Escherichia Coli and by 6 times to the near absence of adhesion for Staphylococcus.Aureus compared to the negative control. In the case of Novocidin, the quantitative result obtained for E.Coli is comparable to that of negative control, while one can observe a strong bacterial adhesion decrease for S.Aureus (about 3 times less than the negative control).

2. In vitro assessment of biocompatibility of materials selected in WP3:
This part is devoted to characterization of cell / biomaterial interactions in static conditions by the evaluation of the cytocompatibility of the scaffolds. The in vitro assessment of the cytocompatibility allows to determine the most relevant proposed materials in order to minimize in vivo studies.
The study consisted of two parts: the first part describes the cytotoxicity study which is the first step in biocompatibility evaluation and the second part describes the cytocompatibility assessment which assess the biological acceptance of the material.
The materials tested are IPNs (interpenetrating polymer networks) described in the work performed in WP1 and WP3, from BioModics and DTI.

2.1. Cytotoxicity testing
The first step in biocompatibility evaluation is the study of cytotoxicity of raw materials or precursor materials in order to eliminate any toxic material at an early stage of the project. Cytotoxicity is assessed according to the standard ISO 10993-5 “Biological evaluation of medical devices- part 5. Tests for in vitro cytotoxicity”.
Acute toxicity causes necrotic cell death, which is assessed with the study of cell viability over 24 hours.

Conclusion :
Of 24 sample materials provided by DTI and BioModics 6 materials have shown cytotoxic effect using established cell line Balb/c 3T3. This allowed to select non cytotoxic materials for further study using human urothelial cells in order to validate cytocompatibility using a reactive cell system from the catheter implantation site in direct contact with the selected materials. The absence of cytotoxicity of materials demonstrates that these test materials will not induce cell suffering and tissue necrosis when implanted in vivo in the ureter or the bladder.

2.2. Cytocompatibility assessment (study of human cell / biomaterial interface)

Based on the results of cytotoxicity testing, the non cytotoxic materials were considered (see above) for cytocompatibility assessment. The goal of this task was to demonstrate the biological acceptance of the material. Two test systems are used: Human urothelial cells and human monocytes-macrophages.

Human urothelial cells:
Among the 15 non cytotoxic test materials 6 test materials allowed more or less HUC adhesion. Of these 6 test materials 3 test materials allowed adhesion and proliferation of HUC. The good quality of the interface between HUC and a sample demonstrates the good cytocompatibility of the test material. However, although the other 12 materials did not allow cell adhesion and growth, they cannot be considered as inappropriate as no cytotoxicity was demonstrated.

Human monocytes-macrophages: Amongst the 15 non cytotoxic materials two materials activated statistically significantly human monocytes-macrophages at 24 h and 48 h assessing IL1-β and TNF-α. They may have some inflammatory potential which is reflected by an increase of IL1-β and TNF-α secretion by human macrophages. All other materials did not activate in vitro significantly human monocytes-macrophages suggesting they have no inflammatory potential in vivo.

In vitro assessment of biocompatibility of selected prototypes: cytotoxicity testing, cytocompatibility testing, genotoxicity testing:

The goal of Task 4.3 is “the assessment of in vitro biocompatibility of prototypes”.
Three aspects are taken into account.
➢ Cytotoxicity testing according to ISO 10993-5 “Biological evaluation of medical devices - Tests for in vitro cytotoxicity” ;
➢ Cytocompatibility testing concerning :
o Human urothelial cells interface with each prototype catheter
o Human monocytes-macrophages activation in contact with each prototype catheter
➢ Genotoxicity testing according to ISO10993-3 "Biological evaluation of medical devices Part 3- Tests for genotoxicity, carcinogenicity and reproductive toxicity"
o Chromosome aberrations in CHO cells (OECD 473 “In vitro Mammalian Chromosome Aberration Test”)
o Micronucleus test in TK6 “human lymphocytes blastoïd cells” (OECD 485 “Genetic toxicology, Mouse Heritable Translocation Assay”)(1)

3. Prototypes tested:

− BioModics IPN
− BioModics IPN with BGs Noble metal coating
− BioModics IPN loaded with Nitrofurazone
− DTI IPN with BGs noble metal coating

Subtask 3.3.1. Cytotoxicity testing

IPN catheters arising from both DTI and BioModics were non-cytotoxic. This test validates the absence of cytotoxic effect of IPN catheters whether they originate from DTI or BioModics. This is a very important point since IPN BacAttack technology is highly innovative and can be the basis of antimicrobial drug impregnation of various catheters and more generally implantable devices.

When coated with BGs “noble metal alloy coating” the original extract is cytotoxic. However, 70 % extracts are no longer cytotoxic. Results obtained with the both materials (DTI IPN BG coated and BioModics IPN BG coated) are superimposable. One can assume that this weak cytotoxic effect (70 % dilution of the extract is not cytotoxic) may be rapidly dissolved in ureteral peripheral tissues and excreted.

BioModics IPN loaded with Nitrofurazone was highly cytotoxic. However, this result demonstrated the loading capacity of IPN with an antimicrobial agent, which is released during extraction, and should be released after implantation. Nitrofurazone has dose-dependent toxicity to human cells (Boyce and al. J. Burn Care Rehabil. Cytotoxicity testing of topical antimicrobial agents on human keratinocytes and fibroblasts for cultured skin grafts 1995; 16 (2 pt 1): 97-103). Here the loading concentration of Nitroflurazone is probably too high and can be optimized for the device.

Subtask 3.3.2. Cytocompatibility testing

Cytocompatibility assessment is the study of the interface between human cells arising from the implantation site and the implanted device.
Two test systems are used:
• human urothelial cells for cytotoxicity and adhesion testing
• human monocytes-macrophages to evaluate in vitro their activation in direct contact with the device.

Studies performed using human urothelial cells

A. Cytotoxicity study (direct contact)
No cell growth inhibition higher than 30 % is measured for IPN and BG-coated IPN from both BioModics and DTI. This demonstrates, according to ISO 10993-5, that the corresponding prototypes are not cytotoxic in direct contact.
B. Adhesion study
− IPN catheters from BioModics and DTI
Results obtained with the 2 IPN catheters are quite comparable to those obtained testing HUC adhesion on the corresponding samples in the framework of selection of the final test material.
This lack of adhesion and proliferation of HUC is not a negative answer of the IPN catheters as it may prevent inflammation and eventual adhesion of the outside of the catheter with the wall of the ureter.
− IPN catheters from BioModics and DTI coated with BG noble metal alloy: No significant adhesion of HUC is observed.

Studies performed using human monocytes-macrophages
No statistically significant increase in cytokine level was observed whatever the prototypes and the cytokine considered (IL-1β or TNF-α). In vitro testing in direct contact with the prototypes did not induce statistically significant activation of human monocyte-macrophages.

Subtask 3.3.3. Genotoxicity assessment

Two in vitro tests have been carried out:
Mammalian Chromosome Aberration to allow the identification of clastogen agents inducing structural chromosome aberrations in metaphase cells. Two kinds of aberration can be induced: chromatid-type aberration and chromosome-type aberration. Both type aberration may not be transmitted to the daughter cells, but the damages may not be compatible with cell survival. Mammalian Chromosome Aberration test performed according to the guideline OECD 473 (short term treatment)

Mammalian Cell Micronucleus to allow the identification of clastogen or aneugen agents inducing micronuclei in the cytoplasm of interphase cells. DNA damages are transmitted to the daughter cells and may not to be compatible with cell survival. Mammalian Cell Micronucleus test performed in the absence of any exogenous metabolizing system according to the guideline OECD n° 487)

Results and conclusion

Mammalian Chromosome Aberration test using CHO cells
This “screening test” (100 karyotypes studied) performed for a short term treatment in both the absence and the presence of a metabolic activation system, let us to conclude that the extracts (100 %) prepared from the 4 prototypes according to ISO 10993-12 “Biological evaluation of medical devices -- Part 12: Sample preparation and reference materials” do not induce chromosome aberrations in CHO cells.

The 4 tested prototypes were found to be non clastogenic in the assay conditions.
This result needs to be confirmed first increasing the number of analyzed karyotypes, second performing the test with a long term treatment (20 h).

Micronucleus test using TK6 cells:
This “screening test” (500 cells observed) performed for short term and long term treatment in the absence of metabolic activation system, let us to conclude that the extracts (100 %) prepared from the 4 prototypes according to ISO 10993-12 “Biological evaluation of medical devices -- Part 12: Sample preparation and reference materials” do not induce in vitro micronucleus in mammalian cells (TK6).

The 4 tested prototypes were found to be non-clastogenic in the assay conditions.
This result needs to be confirmed first increasing the number of cells analyzed, second performing the test in the presence of a metabolic activation system.

General conclusion:
The in vitro biocompatibility assessment of the four main prototoypes taken into account demonstrated:

➢ acceptable cytotoxicity levels for both Balb/c 3T3 and human urothelial cells (HUC), i.e. cells from the implantation site of the device ;
➢ absence of in vitro pro-inflammatory properties using human monocytes-macrophages ;
➢ absence of clastogenic effect (genotoxicity) through chromosome aberration test in mammalian cells (CHO cells) and micronucleus test in human lymphoblastoïd cells (TK6 cells) (screening tests).

These results present an in vitro pre-validation of the biocompatibility of the technology of IPN coated with “noble metal alloys”, or other antibacterial entities in the near future through impregnation loading (nitrofurazone, AMPs,...).

WP5 Microorganism Adhesion, Colonization and Biofilm Formation, Partners SGUL and BG
The main objective of this work package, led by SGUL, was to study the property of the catheter materials (derived from WP3) to assist in the selection of candidate materials for prototype production in WP6. The materials will be tested against microorganism adhesion, colonisation and biofilm formation using microbiological methods. The long-term effects of the coating material such as release will be tested using the same methods.
The bacteria used for this WP will be pathogens which usually associated with CAUTI such as Escherichia coli (NCTC 9434), Pseudomonas aeruginosa (NCTC 6751) and Enterococcus spp. Most importantly, some Gram-negative and Gram-positive bacteria (100 strains) isolated from patients with urinary tract infections and CAUTI will be used to test the clinical significant of these coating materials and final prototype catheters. Also the antimicrobial activities of the materials derived from WP 1, 2 and 3 will be examined.
In WP5 SGUL developed models such as platonic log-phase and stationary phase cultures and biofilm models to test AMP against E. coli and other gram negative bacteria. SGUL also developed a model to measure the constant release of the loading material from the carriers. More than 100 clinical isolates have been characterized.
SGUL tested the IPN discs loaded with AMPs received from the partners applying the disc diffusion method which demonstrated successful testing of material release from IPN material provided partners. The first studies showed that IPN materials loaded with APIs such as nitrofurantoin, trimethoprim, gentamicin, chlorhexidine and Plectasin were effectively released from IPN.
Peptides from NZ showed that Plectasin has activity against Staphylococcus aureus but not E. coli. Novicidin has broad spectrum activity against Gram positive and Gram negative bacteria, but more active against Gram negative bacteria.
9 peptides from WI showed low MIC and bactericidal activities against E. coli and Klebsiella pneumoniae in nutrient broth and in artificial urine.
SGUL has further determined combination effects of a peptide cecropin A with gentamicin, colistin, ceftazidine and cefotaxine. Significant synergy was observed when cecroping A was combined with gentamicin, colistin, ceftazidine and cefotaxime.
Further combinational approaches gave the following results:
1. Nordihydroguaiaretic acid enhances the activities of aminoglycosides against methicillin- sensitive and resistant Staphylococcus aureus in vitro and in vivo
Infections caused by methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and MRSA infections are difficult to treat. There are no new classes of antibiotics produced to the market to treat infections caused by the resistant bacteria. Therefore, using antibiotic enhancers to rescue existing classes of antibiotics is an attractive strategy. Nordihydroguaiaretic acid (NDGA) is an antioxidant compound found in extracts from Larrea Tridentata. It exhibits antimicrobial activity and may target the bacterial cell membrane. Combination efficacies of NDGA with many classes of antibiotics were examined by chequerboard method against 200 clinical isolates of MRSA and MSSA. NDGA in combination with gentamicin, neomycin and tobramycin was examined by time-kill assays. The synergistic combinations of NDGA and aminoglycosides were tested in vivo using a murine skin infection model. Calculations of the fractional inhibitory concentration index (FICI) showed that NDGA when combined with gentamicin, neomycin or tobramycin displayed synergistic activities in more than 98% of MSSA and MRSA, respectively. Time kill analysis demonstrated that NDGA significantly augmented the activities of these aminoglycosides against MRSA and MSSA in vitro and in murine skin infection model. The enhanced activity of NDGA resides on its ability to block the efflux pumps of the bacteria leading to accumulation of the antibiotics inside bacterial cells. We demonstrated that NDGA strongly revived the therapeutic potencies of aminoglycosides in vitro and in vivo. This combinational strategy could be of a great value to contribute a major clinical usage in our fight against antibiotic resistant bacterial infections.
2. Combinations of β-lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus
Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n=101) and MRSA (n=115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin) in 87- 89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We demonstrated that plectasin strongly rejuvenates the therapeutic potencies of existing antibiotics in vitro and in vivo. This is a novel strategy that can have major clinical implications in our fight against bacterial infections.
3. Antimicrobial peptide novicidin synergises with rifampicin, ceftriaxone and ceftazidime against antibiotic-resistant Enterobacteriaceae in vitro
Novicidin, a novel cationic antimicrobial peptide, is effective against Gram-negative bacteria. SGUL investigated novicidin as a possible antibiotic enhancer. The actions of novicidin in combination with rifampicin, ceftriaxone and ceftazidime were investigated against 94 antibiotic resistant clinical Gram-negative isolates and 7 strains expressing New Delhi metallo-β-lactamase-1 (NDM-1). Using the chequerboard method, novicidin combined with rifampicin showed synergy with over 70% of the strains, reducing the minimum inhibitory concentrations (MIC) significantly. The combination of novicidin with ceftriaxone and ceftazidime was synergistic against 89.7% of ceftriaxone-resistant strains and 94.1% of ceftazidime-resistant strains. Synergistic interactions were confirmed using time kill studies with multiple strains. Furthermore, novicidin increased the post-antibiotic effect (PAE) when combined with rifampicin or ceftriaxone. Membrane depolarisation assays revealed that novicidin alters the cytoplasmic membrane potential of Gram-negative bacteria. In vitro toxicology tests showed novicidin to have low haemolytic activity and no detrimental effect on cell cultures. We demonstrated that novicidin strongly rejuvenates the therapeutic potencies of ceftriaxone or ceftazidime against resistant Gram-negative bacteria in vitro. In addition, novicidin boosted the activity of rifampicin.
BG’s activities in this workpackage included:

Field Emission Scanning Electron Microscopy (FE-SEM) analysis:
Hitachi S-4800 FE-SEM (Tokyo, Japan) was used to study the surface changes of the IPN samples. FE-SEM allows for imaging the IPN samples by operating at low voltage with minimum destruction of the soft material. This moreover gives the advantage of low electrical charging and hence possibility of imaging polymers surfaces without conductive coating. After 15 days of soaking in artificial urine, the IPN samples was cut, air dried and kept in desiccator prior to surface analysis. The samples that were exposed to bacteria were analyzed by SEM, in order to examine the surface and investigate the bacterial adhesion.

BG developed and standardized the microbiological Ahearn test, the long term soaking model and the Biofilm formation tube model for the BacAttack samples. Moreover, the remaining silver concentration method on BacAttack samples after soaking as well as the combined methodology to visualize the effect of peptides on Zone of inhibition agar plate test were developed and standardized.

Ahearn test: The primary adhesion of bacteria to the BacAttack-coated and control material were analyzed. The procedure was performed according to Bactiguard Standard Operating Procedures. In the present investigation BG has used bacteria Pseudomonas aeruginosa (ATCC 6751), Escherichia coli (NCTC 9434) and Staphylococcus epidermis
The microbiology studies for the first discs produced from polydimethylsiloxane polymer (PDMS) used for urinary catheters showed that:
• The untreated Polydimethylsiloxane (PDMS) as well as IPN treated PDMS did not reduce bacterial adhesion.
• IPN samples with BG coating showed a high reduction of bacterial adhesion.
• IPN samples loaded with Plectasin showed a time depending release of the peptide from the discs.
• IPN samples loaded with Novocidin and then coated with BG did not release active substance which could be detected in Zone of Inhibition test.
• IPN samples that were first BG coated and thereafter loaded with Novocidin release a discrete antibacterial activity in Zone of Inhibition test.

The prototype was developed using the same PDMS material as is used in BG’s commercially available urinary catheter. The initial colonization and Biofilm formation was measured by microbiological methods such as the Ahearn test model, Zone of Inhibition, and biofilm formation.

The PDMS material was successfully treated by DTI giving a homogeneous IPN with well-characterized properties with respect to swelling and material characteristics.
• DTI used two different AMP´s for the loading of the IPN. i.e. Collistin Sulphate (Sigma Aldrich CAS Number: 1264-72-8) and R4-A (from Peptide 2.0 The loading was successful and the total concentration of loaded substance could be determined. Also the AMP release profile of the material was determined.
• The samples were BG coated according to an optimized method for the IPN materials with a surface concentration of silver and noble metals comparable to those on their commercially available BIP Foley catheters.
• The combination coating and controls were characterized with respect to coating characteristics and tested for ZOI, short and long term antimicrobial effect (adhesion of bacteria), and biofilm formation. The coating had a strong short-term antimicrobial effect towards all strains tested and close to 100 % reduction of E.Coli after 10 days of soak.
• In summary, the prototype tested showed both a short and long term antimicrobial activity, i.e. up to 30 days with a 100 % reduction of bacterial adhesion at all tested time points. However, it is difficult to interpret the results as the control has a 100 % reduction throughout the tests.

WP 6 Prototype and Process Verification
The objective of this work package was to produce a prototype - a urinary catheter with a self-regenerating surface with a two-fold antimicrobial action. The prototype will be characterised using in vitro models simulating clinical use. In vivo and clinical trials will be performed post project as part of BM and BG’s exploitation activities. The partners involved in this work package were BG, BM and DTI. BG led the WP.

Task 6.1 - System Scale-up for Prototype Production
BM successfully scaled the IPN process from 16ml reactors to 2 x 1L reactors. It was found that the batch-to-batch variation and the variation within the batches in terms of hydrogel content are much smaller in the 1L reactor setup than treating small samples in the bench-top 16ml reactors. Having two 1L reactors also resulted in the possibility of treating full sized (already assembled) Foley catheters supplied by BG.
During the initial studies for treatment of full sized assembled catheters it was found that the balloon ruptures during the IPN treatment. BM worked extensively on solving this issue. By adapting the conditions for treatment in the scCO2 reactors and shielding the balloon, it was possible to produce IPN Foley catheters with intact balloons.
DTI’s supercritical system
DTI’s purchased equipment, a state-of-the-art SFE (Supercritical Fluid Extraction) system setup from Waters® with computerised control, was installed in 2012. It is equipped with a reactor/extraction chamber of the size 250 ml, a P-50 pump (max pressure 400 bar), co-solvent pump, ABPR (automated back pressure regulator) and collector vessel. Computerised control of temperature and pressure is monitored via the Process Suite/SuperChrom SFC Suite software. A reactor chamber with window allows for visual inspection during processing. Since 2015, DTI is also equipped with a 1 liter chamber and a P-200 pump with max pressure 685 bar.
DTI implemented the IPN synthesis as a batch process in the SFE system. Each batch is run over a period of 18 hours. The CO2 pump, heat exchanger, reactor and ABPR are monitored continuously. The monitoring of the ABPR allows for visualisation of the depressurisation step during critical time points during the IPN production process.
DTI has collected and analysed the data for evaluation of phase transitions and solubility regimes. The process run has been depicted in a pressure vs. temperature phase diagram. The run is composed of three parts:
1. Pumping of CO2 for pressure build-up in reactor. The pressure data is taken from monitoring the scCO2 pump, showing the pressure build up in the system when liquid CO2 is pumped into the reactor. The procedure takes place during mild heating of the reactor up to a moderate temperature just above the critical point of CO2, meaning that the process conditions remain outside the two-phase gas-liquid region, i.e. phase separation into two phases in the reactor is avoided.

2. Isochoric pressure increase in reactor induced by heating. An isochoric pressure increase (i.e. at constant CO2 density) is induced in the process by heating the reactor. The pump is shut during the heating of the reactor after until a constant set pressure level is achieved. Thereafter the pump is started to adjust the pressure level in the reactor at the maximum pressure and temperature point of the process.

3. Depressurization by the ABPR. The third step in the production route is the depressurization by the ABPR, which implies computerised monitoring of pressure decrease at a controlled rate.

The solubility of CO2 in various selected polymers was simulated by equation of state simulation (simplified Perturbed-Chain Statistical Associating Fluid Theory, sPC-SAFT) and plotted as a function of pressure. The polymers represented in this study were silicone (polydimethylsiloxane, PDMS), polylactic acid (PLA) and polypropylene (PP). The study indicated good correlation between the simulations for the solubility and the experimental data. Results showed that PDMS has the highest CO2 solubility, followed by PLA and PP which has the lowest CO2 solubility.
The above study and evaluation of the control parameters aim at identifying future process controls during a GMP production of catheters.

Task 6.2 - Production of Prototypes and Definition of SOP (BM, DTI, NZ, BG) M34-45
Prototypes produced by BM
More than 80 full size 12 Fr IPN Foley catheter prototypes have been produced by BioModics during the final year of the project. Three different types of prototypes were produced: (i) IPN 30% PHEMA-co-PEGMEA, (ii) Bactiguard coated IPN and (iii) nitrofurazone loaded IPN. In the final 2 months of the project further full sized prototypes were produced: Colistin Sulphate loaded IPN catheters and R4-A loaded IPN catheters.
Prototype produced by DTI
The prototype produced by DTI were IPN-treated catheter sections of approx. 8 cm in length. The prototypes were made from the shaft part of 12Fr uncoated BIP Foley catheters supplied by BG. The hydrogel in DTI’s prototype is the cross-linked poly(ethylene glycol) methacrylate (PEGMA) with OH-terminated group. The hydrogel content of the IPN is 29,2 wt% with a SD of 1,76 wt%.
DTI has sent IPN-treated catheter sections for coating by BG followed by ETO sterilisation. Sterilised IPN-treated catheter parts with and without Bactiguard coating were sent to LEMI for biocompatibility testing as reported under WP4.
Selected prototypes were sent to project partners for the following testing: Ahearn tests by BG, biocompatibility testing by LEMI, AMP release test at WI and Antimicrobial studies by SGUL.
An essential activity for sample and prototype development is definition and regular updating of Standard Operating Procedures (SOPs). Both BM and DTI have registered their SOP’s for future manufacturing of prototypes. SOP’s have also been generated for discs produced under the conditions for prototypes as these were to be used for in vitro biocompatibility testing under WP4.
Loading of antimicrobial substances
IPN materials have been loaded with antimicrobial substances during the course of the project by soaking in a loading solution. The loading solution was prepared by dissolving the antimicrobial substance in an appropriate solvent. SOP’s for different active pharmaceutical ingredients are also registered.
Task 6.3 - Characterisation of Final Device
Swelling measurements of DTI’s prototype: The IPN-treated 12Fr catheter tubes were analysed by swelling measurements in different media: urea broth (Sigma Aldrich), saline solution (0,9 wt% NaCl) and demineralized water. The prototype increases in weight by approximately 50 wt% during soaking in urea broth, saline solution and demineralized water. After 24 h, maximum swelling has occurred.
MicroCT scanning of DTI’s prototype
Micro-CT scanning was used for the characterization and evaluation of the homogeneity of the IPN-treated catheter section. The scanning was carried out to assess cross sectional changes of silicone catheter sections prior and post IPN treatment, as well as after soaking in urea broth for 3 days. Results showed that the IPN treatment and the soaking catheter does not block the catheter. The inner diameter and wall thickness increase to the same extent indicating a homogenous treatment.
Shelf life and ageing of prototype
Mechanical testing was performed on silicone catheter tubes and DTI’s prototype after exposure in ageing tests.
Series 1 was exposed to ageing in simulated physiological conditions in a urine flow system at 37°C that had been specially developed by the DTI. The exposure times for the study were 0 (start), 1, 2 and 4 weeks.
Series 2 was exposed to accelerated ageing in oven at 60°C over a period of four weeks. The selected time points were 0 (start), 1, 2 and 4 weeks (end). The accelerated ageing tests were performed according to ASTM F1980 “Standard Guide for Accelerated Aging of Sterile Barrier Systems for Medical Devices” and represented a shelf life of one year.
Mechanical testing
The methods for the tensile testing were performed with reference to standards ISO527 “Determination of tensile properties” and ASTM D 638 “Standard Test Method for Tensile Properties of Plastics“. The standard methods for mechanical testing of full-length urinary catheters, i.e. EN 1616 “Sterile urethral catheters for single use” and 1618 “Catheters other than intravascular catheters – Test methods for common properties” were not used for these samples due to the limited length of the catheter tube parts (length 7-8 cm).
At the selected time points, triplicates of catheter tubes were removed from the exposure test and a range of mechanical parameters were determined by tensile testing. The stress (MPa), strain (%) and E-modulus (MPa) were measured.
The results of the Tensile tests for Series 1 under simulated physiological conditions showed little change in the E-modulus over the 4 week period indicating no degradation of mechanical properties during the expected use of 4 weeks of the catheter.
The results of shelf life studies assessed by accelerated ageing of DTI’s prototype, Series 2 showed that whilst there was little change, as expected, in the mechanical properties of the standard silicone catheter, there is a significant impact on the stability of the IPN materials under the chosen conditions. One should therefore consider whether, in relation to the stability of the hydrogel, a temperature of 60°C is suitable for accelerated ageing.
Optimisation of Bactiguard’s coating of IPN treated catheters:
Bactiguard’s (BG) main objective of this work package was to be part of the development of a final prototype for the BacAttack project. The prototype is a silicone material treated with a combination of the supercritical CO2 (scCO2)/Interpenetrating Polymer Network (IPN) technology, with/without loaded antimicrobial AMP´s, and the BG coating. This is in line with the final goal for the BacAttack project.
The BG coating is used commercially on silicone catheters. It has been well documented for being effective in reducing the adhesion of microorganisms on coated material surfaces such as the BIP Foley urinary catheter. The aim with the prototype is to enhance and prolong this effect by the release of AMP that is loaded into an IPN treated silicone catheter. Two antimicrobial peptides (AMP´s) were loaded and tested, i.e. R4 and Colistin Sulphate (CS).
As a first step, an optimisation of the BG coating was carried out. Initial procedures using the SOP for coating silicone catheters indicated a higher level of silver ions on the surface of IPN treated silicone catheters. In the early stages of the project, the BG coating and metal release were adjusted in order to mimic the commercial available product, the BIP Foley. The silver surface concentration for the final prototype was determined using Atomic Absorption Spectroscopy (AAS) analysis (Air-Acetylene flame) according to BG’s SOPs. Results showed a surface concentration of 2.0 to 2.5 micrograms/cm2 and with a release that is in the same range as for the commercial available BIP Foley catheter.
Furthermore, the prototype material was tested for AMP loading, release profile of AMP, microorganism adhesion, colonization and biofilm formation. Short- and long-term antimicrobial effects were tested and reported. The long-term antimicrobial effect was tested using the standard Ahearn method used by BG. The bacteria used for this was a pathogen, that is highly associated with CAUTI, i.e. Escherichia coli (NCTC 9434) and the data is reported under WP5.
Methodology involved a cumulative soaking procedure of the samples with 2 ml of distilled water and incubated at 1 day, 3 days, 5 days, 10 days and 30 days. The effect of antimicrobial capacity released from BacAttack samples was analyzed using Houlton agar plate cultured with E coli. 10 ul of soaked solution was seeding on agar plate, incubated 24 hours in order to visualize the Zone of Inhibition produced.
In conclusion, we have demonstrated that a catheter material can successfully be treated with IPN and loaded with various AMP’s and subsequently also be coated with the Bactiguard coating. The released AMP’s from the treated material show antimicrobial activity. An IPN treated material formulated by DTI without AMP loading nor BG coating also shows a significant reduction of bacterial adhesion. Towards the end of the project we managed to produce full-length catheters treated with IPN, loaded with AMP’s and coated with the Bactiguard coating which have shown strong reduction of bacteria, up to 100% for the timeframe for the studies. Due to lack of time, the final prototypes containing the R4 and CS AMPs were not evaluated. However, the indications are that the antimicrobial properties should be at least equal to the prototypes tested.
Finally, we can conclude that we have achieved the goals and objectives of the BacAttack project.

Potential Impact:

Potential impact of the results of the BacAttack project

Strategic impact
Bacterial growth on medical devices may be prevented by immersing the medical device in a coating mixture containing an antimicrobial agent prior to its use. Unfortunately, it is difficult to attach a surface coating on silicone due to its hydrophobic nature and excess of oils migrating to the surface. In addition, such surface treatments, often have a short-lived effect due to the limited amount of antimicrobial agent in the thin surface coating and the loss of coating by physical wear, with one exception being Bactiguard’s patented noble metal coating.

Alternatively, patients can receive systemic treatments with antibiotics in huge amounts with risk of bacterial resistance and impaired health of the patient and damage to the environment. The outcome of the BacAttack project will avoid the above problems through a constant dosing of antimicrobial agents for a long time which is only applied locally. The procedure is non-invasive because the agents are fixed to the catheter (Bactiguard) or released just prior to function and subsequent local degradation (AMPs). The hybrid material regulating these properties is also non-invasive due to its composition of inert silicone rubber carrying an inert hydrogel network. The outputs from the project will significantly impact European quality of life with a hybrid material solution preventing infections, saving cost and improving patient management. The most appealing perspective might be the ease of transferring the post-treatment of polymers to many different devices providing a broad industrial and societal effect. Effective local treatment will result in fewer side effects, better primary effects and easier drug administration compared to systemic treatments. In addition, novel biomedical devices developed here will improve Europe’s competitiveness and innovation.

The BacAttack project contributes to the specific impacts outlined in the HEALTH 2011 2.3.1-5 Development of tools to control microbial biofilms with relevance to clinical drug resistance in the following way:

1. Availability of tools that control, disrupt or prevent biofilm formation:
• New IPN technology allowing local treatment with unique long term and controlled release. The silicone IPN drug delivery concept has the potential to improve and/or replace low performing material coatings, risks related to systemically introduced nanoparticles and unsuccessful implants.
• Two-fold antimicrobial strategy of antimicrobial peptides (AMPs) and metal alloy.
• Broad spectrum AMPs to treat various bacteria and fungi.
2. Better management and improved treatment of infections caused by pathogenic bacteria and fungi.
• Long term antimicrobial effect gives longer lifetime of catheters.
• Reduced patient morbidity.
• Fewer/no side effects and a better quality of life.
• Strong society impact due to better drug administration. Known drugs can be applied more efficiently.
• Exposed groups – children and elderly - can receive treatments which are better tolerated and with a higher degree of reliability.
• Avoiding non-compliance issues as experienced with systemically applied drugs – particularly relevant for children and elderly patients who are under the care of untrained healthcare workers.
• Health care providers e.g. hospitals can effectively reduce costs relating to infection management using these systems.
• Add-on technology to existing medical devices.
• Technology applicable to various materials.

Impact on the Health of European Citizens
The increased prevalence of antibiotic resistant bacteria poses a serious threat to health of European citizens, with cases of hospital acquired UTI’s in the five largest Europe nations exceeding a total of 1.5 billion cases per year (Hospital-acquired Infections – Trends Across Europe’, Frost & Sullivan, June 2010). The European Centre for Disease Prevention and Control (ECDC) has estimated that the annual number of infections caused by antibiotic resistant bacteria is 386 100 resulting in 25 100 deaths (two-thirds from Gram-negative bacteria), with 37 000 patient deaths as a direct consequence of HAIs and an additional 111 000 as an indirect consequence of HAI (Tacconelli et al, J. Hosp. Infec. 72(2) (2009) 97-103). Non fatal infections resulted in patients spending an extra 2.536 million days in hospital. Collectively the direct and indirect effects of HAIs for the EU have been estimated as EUR 1.534 billion per annum.
According to the World Health Organization (WHO) estimates, presently, over 200 million people are suffering from one or the other bladder control problems that essentially require urinary catheterization. The National Health Service (NHS) of the U.K. estimates that in 2013, approximately 3 million to 6 million people were suffering from some degree of urinary incontinence in the UK. Globally, a large number of patients would require administration of urinary catheters at one or the other instance during their lifetime. As the average lifespan increases in major regions of the world, the numbers in the patient pool requiring urinary catheters will increase. This in turn will contribute in the future to rapidly increasing the revenues generated by urinary catheters.

The present state-of-the-art coatings for catheters are inadequate because of their fragility, poor release profiles and short life time. In a recent study of catheter related bloodstream infections (CRBSI) in four European countries (France, Germany, Italy and the UK) it was reported that the incidence of CRBSIs in these countries lay between 8 400 – 14 400 episodes/year/country and that the direct costs to these countries of CRBSIs was between EUR 35.9 million and EUR 163.9 million annually(Tacconelli et al, J. Hosp. Infec., 72(2) (2009) 97-103). Using figures drawn from the study it has been estimated that the annual costs of CRBSIs to the EU is currently around EUR 1 billion and increasing as bacteria become more antibiotic resistant. Indirect costs based on the number of CRBSIs/country/year are estimated to be at least as large as the direct costs.

Commercial Impact of BacAttack:
According to a new market report published by Persistence Market Research ( ): “The urinary catheter market accounted for 5.1% of global catheter market in 2014 and was valued at US$ 1,326.0 million. Revenue contribution by urinary catheter market to the global catheter market is expected to increase to US$ 1,755.0 by the end of 2021, expanding at a Compound Annual Growth Rate (CAGR) of 4.1% between 2015 and 2021. The increasing prevalence of chronic diseases and growing ageing population are factors expected to positively affect the growth of the urinary catheter market across the globe.”

However, in a global perspective, the health care sector is facing a problem with no effective solutions for combating resistant bacteria. No tool is available to deal with the problem in an effective way. Therefore the strategy of local prevention and combination strategies appears to be very promising. The growth in medical devices related to these problems is estimated to be 40% each year. At the same time medical device coatings is the most rapidly expanding sector of the EUR 220 billion global medical device market (of which the EU accounts of approximately a third with total sales of EUR 72.6 billion in 2007)(About the Medical Technology Industry’, Eucomed Website, accessed 7 Feb 2011). These coatings increase the functionality, longevity and cost-effectiveness of medical devices. However, the whole concept of using antibacterial coatings on medical devices has failed to a large extent due to poor release profiles, because the coatings are thin and fragile. This leaves an empty gap in a market which is in urgent need of a better solution and differentiated products. The solution outlined in this project will be to make a robust surface which is supplied with active components from a depot within the material.

In North America alone more than 100 million urinary tract devices including urethral catheters, ureteral stents and penile prostheses are inserted each year, which results in millions of device-related infections and billions of dollars in additional healthcare expenditure each year. These devices provide novel, non-host surfaces on which bacteria can colonise and form biofilms. Short term use of urethral catheters, for example, during surgery to monitor urinary output, does not typically result in urinary tract infections, because there is insufficient time for the bacteria to colonise the catheter and form a biofilm. However, in patients undergoing long-term catheterisation because of urine retention or incontinence, recurrent urinary tract infections are common. The risk of infection is related to the length of time the catheter is in situ. The major bacterial species involved in long term catheter and stent infections are Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus epidermidis, Enterococcus faecalis and Staphylococcus aureus. These organisms are commonly isolated from the patient’s gastrointestinal tract, thus providing a constant source of bacteria for re-infection if a device is changed or the patient undergoes a course of antibiotics to clear the infection.

Improvement in available urinary catheters and incoming of new and technologically advanced and effective antimicrobial urinary catheters in future would bring future growth opportunities for this market.

Rapid strides have been made in the area of novel drug delivery systems (NDDSs) during the last couple of decades, which has highlighted the importance of intellectual property rights (IPRs). Recently, a large number of NDDSs have been introduced that offer a high degree of therapeutic efficacy and patient compliance, and widen the market share of dosage forms for existing drug molecules. The complexities involved in NDDS design makes IP issues of paramount importance.

The main result of the proposed work is the establishment of an innovative technology platform for drug delivery based on the synergy from merging novel polymer materials with advanced drug loading technology. Due to thorough market analysis and close proximity to industrial key players this technology platform will meet an existing demand for new and better medico-technical devices, but also open up for hitherto unseen drug delivery methods. The IPN drug delivery concept has the potential to improve and/or replace low performing material coatings, risky nanoparticles and unsuccessful implants. New treatments can become available due to the high degree of safety and performance of the drug delivery. Common to all these applications are an increased welfare for patients and other users. They will benefit from better treatments such as long lasting implants and better and longer lasting dosing of their medication. They will have fewer side effects and a better quality of life. This will be combined with a non-invasive (no needles) approach and will be of particular importance for children and elder citizens.

The expected market share for catheters based on BacAttack developments is expected to be around 10% generally due to a new legislation in the USA requiring hospitals to pay for catheter mediated infections themselves. The cost is of the order of EUR 37 000 per patient providing a strong motivation to buy the new catheters. This will be accomplished through collaboration with a major commercial player in the market. Related segments will develop into competitive products over time. Some of these segments (venous catheters) can be covered by a 100% market share as effective alternatives do not exist. Furthermore bloodstream infections have a much higher frequency compared to UTIs.

The European medical devices industry employs 529 000 people in approximately 11 000 companies of which 80% are SMEs and invests EUR 5.8 billion in R&D annually The medical device sector is increasingly focusing on the use of combination products involving ‘device and drug’. Whilst the EU has a strong foothold in medical device technologies, it is under increasing threat from manufacturers in technologically advanced countries such as Japan and, in particular the USA, driven by their national legislation and health insurance requirements, aging population problem and the spread of multidrug bacterial resistance.

By developing new antibacterial materials for incorporation into medical devices such as catheters and stents, the BacAttack project will help to support the continued growth in the SME dominated European medical device and health care sectors, protecting existing jobs as well helping to generate new employment opportunities in a high technology, knowledge based industry.

Other Applications for the BacAttack Strategy
The IPN-concept opens the possibility to vary many parameters in order to optimize the release profile from the IPN to suit the characteristics of the drug compound, e.g. solubility, bulkiness, sensitivity to enzymatic degradation etc. Relevant parameters are the polymer morphology, production techniques and macroscopic shape of the drug delivery system. This makes the technology suitable for application to other medical devices than catheters, such as stents and wound care products. IPN hybrid materials can be used for almost all tubings and devices used in hospitals, medical centres and nursing homes.

Environmental Benefits
The BacAttack processing technologies used for producing the IPN based materials offer significant reductions in detrimental environmental impact. By utilising on-line process control the material and energy consumption is optimised and the active drugs are handled in a closed pressurised system allowing optimal waste handling and recycling. The finished products can be disposed of easily in contrast to drugs with no delivery systems. The lack of drug delivery systems can result in increased use of high doses of systematic treatments and subsequent exposure to the environment. In relation to bacteria and antibiotics, this may result in increased levels of multidrug resistance. Therefore, IPNs are of great benefit to the environment. Even the fundamentals of the production are green as CO2 replaces dangerous organic solvents in the impregnation steps.

Steps Needed to Bring About the Expected Impacts
The focus of the present project was to implement new surface treatments for clinical applications. The combination of modern biotechnology with advanced polymer chemistry/nanotechnology has the potential to cause a paradigm shift in non-invasive drug delivery and new treatments. The steps to bring about the impacts included:

1) A prototype device in the form of a catheter manufactured using the new IPN technology.
2) In-vitro assessment. The performance of the prototype device be tested and bench-marked against state-of-the-art technologies.
3) Commercialization. Bactiguard is a worldwide market-leader within antimicrobial solutions for catheters and a provider of anti-infectious coatings and would, therefore, be ideally positioned to rapidly bring the new IPN-based product to market.
4) Regulatory issues. All materials are already FDA-approved (EU class 2). The rest of the process will be based on a rapid 510k approval. The new AMPs will need to go through vigorous testing before they can be applied. However, based on the outcome of this project other APIs can be effectively applied.
5) Dissemination. Reports describing the in vitro assessments will be published in scientific and trade literature, and presented at conferences and trade fairs.

Of these steps, those related to obtaining certification of devices based on the BacAttack technology are vital. To address these steps, Biomodics and Bactiguard had proposed, post project, to organise and supervise in vivo assessments and clinical trials of the BacAttack technology and prototype as part of their post project exploitation activities. With all medical devices, meeting the regulatory requirements is paramount. The prototype device that has been developed is a borderline device, i.e. a medical device incorporating as an integral part an ancillary antibacterial medicinal substance. Hence, our work plan includes tasks to specifically address the requirements for achieving CE certification of the final catheter based on the prototype catheter developed in the BacAttack project.

Rational for a European Approach to achieve the impacts
The BacAttack project involved the development of a new tool for minimizing drug resistance by upgrading existing and future medical devices through a unique self-regenerating surface that prevents biofilm formation by a two-fold stealth attack. This required the combination of developments in both materials science (polymers, metallic coatings and scCO2 processing) and the life sciences (antimicrobial peptides) coupled with an understanding of the clinical requirements and patient management. This was required to unleash the full potential of each technology in an unusually high material/drug synergy. No single member state possesses the institutions and industrial organisations with the specific capabilities capable to achieve the project developments. Thus we brought together a transnational consortium drawn from leading public and private organisations from academia and industry.

A key focus was to assist in developing the European Research Area by bringing together leading European actors in the R&D and exploitation of biomedical materials and devices, contributing to making Europe a global Centre of Excellence within this area and assisting in achieving the objective of the Lisbon Treaty of making the EU the world’s leading knowledge-based economy. Our project will contribute to the ECs objective ‘to secure world excellence in basic research’(Political Guidelines for the New Commission’, J M Barroso, EC, 2009) through large-scale collaborative research.

External Factors that will determine whether Impacts are Achieved
As mentioned earlier, WHO and the European Centre of Disease Prevention and Control has stated that there is a very pressing need to combat antimicrobial resistance which is one of the main objectives of the BacAttack project. However, to compete in this arena the partners must be able to produce competitively priced devices and demonstrate the value that their combination of drugs and delivery systems brings to the market.

The pressing need to resolve HAI has resulted in new legislation in the USA which has forced hospitals to cover many of the costs related to HAI – including those related to CAUTI. Implementation of legislation in hospitals has accelerated the adoption of combination devices. The USA is now a forerunner in implementing strategies for combating bacterial/fungal infections.

The EU and the rest of the world are also trying to address HAI issues and it is expected that this will lead to further legislation. Approval mechanisms to allow the use of combination medical devices within the EU are presently being refined to support this trend. The legislation in the USA has created a new technology scene aimed at addressing the problems created by HAI. Europe could become a key player in this new technology scene. However, at present, few European HAI initiatives are related to new procedures and follow up technologies. All Europe healthcare authorities have to meet stringent budgetary targets however, because of the fragmented nature of many authorities, there is sometimes a lack of a holistic socio–economic approach to healthcare problems. In the case of HAI the cost saving achieved by using cheap latex based catheters are not always balanced against costs related to the fact that such catheters are allergenic and infection prone and can result in serious infection requiring a longer, more costly, stay in a hospital. European legislation in this area could be used to encourage authorities to take a more holistic approach. If present trends continue there seems little doubt that problems related to HAI will have an ever increasing detrimental influence on European healthcare systems. This could ultimately see European legislators being forced to introduce the same type of legislation that has already been adopted in the USA. If European researchers do not develop new devices and technologies to address the problems of HAI in the near future, legislation in Europe, introduced at some later date, could actually force European healthcare authorities to accept US standards and products.

Dissemination of the Knowledge and Results
During the course of the project, there has been extensive dissemination of knowledge and results in the form of peer reviewed articles and presentations at conferences, exhibitions and tradeshows.

Exhibitions, Conferences, and Tradeshows
It is the intention of the BacAttack consortium to continue actively in promoting the BacAttack technology by attending exhibitions and conferences addressing the medical devices and healthcare industries. The industrial partners will use exhibition stands at national and international tradeshows to promote the BacAttack developments to industry and end users whilst the research partners will deliver presentations at conference describing their activities and results to the scientific community. During the timeframe of the project a project website was established which allows for public access to the principles of and non- sensitive activities within the project. There has been activity at 10 exhibitions, 6 poster presentations at scientific events, 4 publications in the popular press, organisation of 3 conferences and 1 workshop, oral presentations at 10 scientific events and events addressing a broader audience. Furthermore, 1 flyer for the BacAttack project has been produced for distribution to enhance awareness of the activities of the project. A video for demonstrating the IPN technology has also been produced that can be used as a marketing as well as education tool.

Articles in Peer Reviewed Journals
In order to disseminate the BacAttack project results into the scientific community the research organisations and universities in the consortium will author scientific articles describing the scientific and technical achievements of the BacAttack project for publication in peer-reviewed journals. During the course of the project partners have published 7 peer reviewed articles in high impact journals including Nature Communications and Frontiers in Microbiology. Further articles are expected to be published after the end of the project.

Publications in Industrial Journals/Magazines
In order to stimulate interest in the commercial take up of the BacAttack developments within the medical devices industrial sector the project’s industrial partners will continue the production of articles for publication in trade journals relevant to the BacAttack developments. Partners will also continue promotion through Professional and European Organisations. The BacAttack consortium partners will use professional bodies (and their ‘trade’ publications), networks, European Organisations, trade associations and European Technology Platforms (ETPs) and COST actions such as iPROMEDAI as a means to disseminate the results and benefits of the BacAttack project.

Training Activities
To ensure the rapid commercialisation of the BacAttack technology, European manufacturers of medical devices will need to be educated in the handling of the new materials and in their quality control. It is expected that the demand for these training courses will increase as the materials start to penetrate the market and will be offered after the completion of the project under normal commercial terms. They will cover the new technology and its application.

List of Websites:

Coordinating partner:
Naseem Theilgaard
Teknologisk Institut, Plastteknologi
Gregersensvej 7, DK-2630 Taastrup